The present invention relates to a novel method for the preparation of nanoparticles formed from a polymerised methylidene malonate compound, said nanoparticles, optionally containing one or more biologically active molecules, as well as to pharmaceutical compositions containing them.
"Nanoparticles" is understood as meaning sub-micron particles having a diameter of less than about 500 nanometres. Nanoparticles formed by emulsion polymerisation of an alkyl cyanoacrylate are described in the EP 0 007 895 patent. The method used in the preparation of these alkyl cyanoacrylate particles relies on the (anionic) polymerisation of the monomer which takes place spontaneously and in an aqueous medium. The preparation which follows the same principle (anionic emulsion polymerisation) of nanoparticles constituted of a methylidene malonate polymer is described notably in F. Lescure et al, Pharm. Res., 1994.11L 1270-1276. These monomers, whose preparation is described in the EP 0 283 364 patent, have a structure close to that of the cyanoacrylates but the nitrile function of the latter is replaced with an ester or an ester ester. Like the cyanoacrylates, they polymerise in the cold in an aqueous medium and can be biodegradable.
However, the methylidene malonate nanoparticles thus obtained possess certain drawbacks.
In fact, the emulsion polymerisation of methylidene malonates in the form of nanoparticles leads, in aqueous phase and at slightly acid pH, to the formation of oligomers, mainly of the trimer or tetramer type, which are highly biodegradable.
These molecular species are partially hydrosoluble, such that the dispersion of these nanoparticles in an aqueous medium leads to their solubilisation and to the rapid loss of the particle structure (P. Breton et al., Eur. J. Pharm. Biopharm., 1996, 47, 95-103). When a biologically active molecule is associated with the methylidene malonate nanoparticles, is therefore possible for the molecule to be released very rapidly after the administration, following the effect of dilution in the circulatory current which brings about the rapid solubilisation of the oligomers which form the particle matrix, before eventually arriving at the site of action of the active principle.
Certain experiments have shown that the polymerisation at basic pH enabled the formation of polymers of higher molecular masses while maintaining the size of the nanoparticles. However, such syntheses are characterised by:
the impossibility of obtaining polymers of Mw&lt;10000, and a fortiori Mw&lt;8000, constituting individualised nanoparticles without forming aggregates and without the significant presence of oligomeric species. PA1 the impossibility of constituting polymers of Mw&gt;20000 and a fortiori of higher Mw, at high pH (pH&gt;7) without the inevitable formation of aggregates which render the intravascular administration of these preparations impossible. PA1 polymers of average molecular mass between about 5000 and 10000, notably about 8000, PA1 polymers of average molecular mass greater than 20000, without forming aggregates. PA1 it enables a more homogeneous dispersion of the monomer in the polymerisation medium, PA1 it makes use of non-chlorinated solvents which are easy to evaporate since they are volatile, PA1 it prevents the formation of polymer aggregates, PA1 it gives rise to high polymerisation yields PA1 it enables the constitution of polymers of homogeneous wide-ranging molecular mass (Mw about 2000 to 100000, notably about 2000 to 80000) in forming nanoparticles having a diameter of less than 500 nm. PA1 the monomer concentration in the organic phase, PA1 the pH and the molarity of the polymerisation medium, PA1 the nature and the concentration of the dispersing agent, PA1 the volume ratio of the aqueous phase (polymerisation medium)/organic phase, PA1 the mode of introduction of the organic mixture in the aqueous phase. PA1 A represents a ##STR5## PA1 group or a ##STR6## PA1 group; PA1 R.sub.1 and R.sub.2, identical or different, represent a linear or branched C.sub.1 -C.sub.6 alkyl group; PA1 n=1, 2, 3, 4 or 5; PA1 characterised in that the monomer(s) is (are), before the polymerisation. dissolved in a water-miscible aprotic organic solvent forming, with the polymerisation medium, a non-solvent mixture of the polymer formed. PA1 A represents a ##STR8## PA1 group or a ##STR9## PA1 group; PA1 R.sub.1 and R.sub.2, identical or different, represent a linear or branched C.sub.1 -C.sub.6 alkyl group; n=1, 2, 3, 4 or 5; PA1 preparing a solution of at least one compound of formula (I) in a water-miscible aprotic organic solvent. PA1 adding, with stirring, this organic phase to an aqueous polymerisation medium at a pH between 4.5 and 10, PA1 recovering the nanoparticles thus obtained after homogenisation of the mixture and evaporating the organic solvent in vacuo. PA1 preparing a solution of at least one compound of formula (1) in a water-miscible aprotic organic solvent, PA1 adding, with stirring, to this organic phase an aqueous polymerisation medium at a pH between 4.5 and 10, PA1 recovering the nanoparticles thus obtained after homogenisation of the mixture and evaporating the organic solvent in vacuo. PA1 the concentration of monomer(s) of formula (I) in the organic solvent is of the order 30 mg/ml to 150 mg/ml; PA1 the molarity of the polymerisation medium is of the order of 1/30 M to PA1 volume ratio of the aqueous phase to the organic phase is between 3/1 and 20/1, preferably between 3/1 and 15/1.
"Mw" is understood as meaning the mass average molecular mass (or average molecular mass) defined as: Mw=.SIGMA.ni. Mi.sup.2 /.SIGMA.ni. Mi and Mp means the molecular mass of the quantitatively major species.
In the rest of the description, the molecular mass is expressed in polystyrene equivalents (Ep).
This preparative method is therefore not suitable if it is desired to prepare methylidene malonate nanoparticles constituted of: